Circadian clock disruption promotes the degeneration of dopaminergic neurons in male Drosophila

Sleep and circadian rhythm disruptions are frequent comorbidities of Parkinson’s disease (PD), a disorder characterized by the progressive loss of dopaminergic (DA) neurons in the substantia nigra. However, the causal role of circadian clocks in the degenerative process remains uncertain. We demonstrated here that circadian clocks regulate the rhythmicity and magnitude of the vulnerability of DA neurons to oxidative stress in male Drosophila. Circadian pacemaker neurons are presynaptic to a subset of DA neurons and rhythmically modulate their susceptibility to degeneration. The arrhythmic period (per) gene null mutation exacerbates the age-dependent loss of DA neurons and, in combination with brief oxidative stress, causes premature animal death. These findings suggest that circadian clock disruption promotes dopaminergic neurodegeneration.


H2O2 treatment.
Minor: 1) Line 74, I would use plural for the MBs (bodies instead of body).
2) Line 89: Writing that PAM-a1 are "selectively" susceptible might be too strong since two other groups of PAM neuron show some sensitivity.3) Rephrase line 146. 4) Line 172-175: Why do flies drink so much more in DD? 5) Paragraph starting at line 197: I presume the clkGAL4 driver used here is also expressed in glia and other tissues.I understand that later the authors focus on circadian neurons, but I would suggest mentioning in this paragraph that the driver is not just expressed in clock neurons, and modifying the conclusion on line 204, 6) Line 245: when introducing split-GAL4 drivers, it would be worth indicating which regions of the MB each innervate.7) Line 286: Ca2+ peaks occur at CT5 and CT17.They would thus be better described as "just before midday and midnight", rather than "morning and early night" 8) Line 287: in the sentence "Because Ca2+ levels in neuronal cell bodies can be correlated with firing frequency, we wanted to know whether decreased excitability would be neuroprotective" is unclear.This does not seem to flow logically: how does the association between firing frequency and Ca2+ levels lead to questioning if excitability was associated with increased vulnerability? 9) Figure 2A/B: Is the "number of PAM neurons," represented in 2B, cells that co-stain with Red Stinger and GFP, or just Red Stinger-positive cells?Also, the representative images chosen for the per0 brains in 2A do not seem to correlate or represent well the data presented in 2B.In particular, the number of RedStinger-positive cells at Day 1 seems to be much lower than at day 7 or day 14.I must say the image quality is not very good in the file we received, so perhaps some of the staining is obscured by the poor resolution.10) Figure 4D: although the PDF-5th sLNv is included in the text as one of the neurons that could project to the PAM neurons, the diagram does not depict it sending projections to the PAM neurons.11) Figure 5B: MB441B should be moved to the rightmost panel (switched with MB315C) to match the order in which these drivers are introduced in the text.12) Figure 5.It would be preferable to keep the order of GAL4 and split-GAL4 drivers consistent throughout the figure.13) Figure 5E: the representative images do not correlate with the quantification in 5F.For the images depicting H2O2 exposure at ZT8 and ZT20, it appears that both have about 8-10 neurons left, in contrast with 5F.
Reviewer #2 (Remarks to the Author): The study by Dorcikova et al. investigates whether Parkinson's disease (PD) is associated only with disturbances in sleep and circadian rhythms or whether these disturbances are causally responsible for PD, as they often precede the actual PD symptoms.To answer this question the authors used the well-established model Drosophila melanogaster, in which the PD typical loss of dopaminergic (DA) neurons can be induced by oxidative stress (e.g.feeding the flies H2O2).The group of Emi Nagoshi has previously shown that H2O2 feeding causes the loss of specific DA neurons in the PAM cluster and that this leads to defective climbing behavior suggesting a partial analogy between the PAM cluster and the mammalian substantia nigra.The PAM neurons comprise ~130 quite heterogenous neurons that, in addition to locomotion, play critical roles in olfactory associative learning, foraging and sleep.
To test for circadian rhythms in vulnerabilty to oxidative stress, the authors developed a protocol for short-term H2O2 treatment (4h of 5% or 10% H2O2) that can be administered at different times of day.They show that this treatment leads reliably to the loss of specific PAM neurons and that the loss depended on the time of day.More importantly, they demonstrate that the rhythm in vulnerability to oxidative stress is controlled by the circadian clock since it persists in the absence of external Zeitgebers and is absent in arrhythmic per0 mutants.Moreover, they show that arrhythmic mutants (per0 and tim0) possess fewer DA neurons in the PAM cluster even without H2O2 treatment and that they loose more DA neurons in response to oxidative stress.Thus, the circadian clock controls the magnitude and temporal sensitivity of the PAM neurons to oxidative stress.Subsequently, the authors performed several critical control experiments that confirmed that it is indeed the circadian clock in specific clock neurons (the PDF-positive LNvs) that regulates the survival of PAM neurons after oxidative stress.Furthermore, they identified the specific PAM neurons (PAM-e1) that were predominantly killed by oxidative stress and showed that the LNd clock neurons are presynaptic to them.They demonstrate that the PAM-e1 neurons show bimodal rhythms in intracellular Ca2+, which are absent in per0 mutants and show that the loss of these neurons affects sleep but not climbing behavior.Finally, the authors investigated the interplay between genetic factors, environmental risk factors and age on the loss of DA neurons in the PAM cluster and found that all factors contribute.This confirms the multi-hit hypothesis for developing PD.
Overall, the study is carefully conducted and clearly shows that disruption of the circadian clock promotes dopaminergic neurodegeneration in flies.The results obtained suggest that mutations in circadian clock genes are an additional risk factor for the development of PD, along with environmental factors and age.These findings could also be relevant for humans and significantly expand our knowledge of the causes of PD.
Nevertheless, the study has several weaknesses that need to be improved before publication.In some places, relevant literature is not cited or discussed and alternative hypotheses are not mentioned.Furthermore, the methods used are not always adequately described.Lines 139-143, Figure 2 and Material and Methods: w1118 and CantonS (CS) flies were used as controls for per0 flies, but none of these stocks is listed in M & M. I suppose that per0 flies are red eyed, thus CS flies are probably the better controls.Do you have an explanation for why the w1118 flies are as sensitive to 10% H2O2 as the per0 mutants (Fig. 2D)? Figure 6 and Material and Methods: I did not completely understand how you generated Figure 6C and D. As you stated in Material & Methods Ca2+ imaging could be performed in a single fly for up to 6-8 hours and imaging was started every 6 hours to cover the entire day.How many flies could you record at once, and how many cells you imaged on average in each single fly?How similar were the fluorescence intensity values in different PAM neurons within the same fly?How did you detect the death of a fly, and how did you decide when to stop measurements on individual flies and discard the data?If I understood it right, at certain time points, the diagram for per0 mutants includes the measurements of only 2 flies.At how many time points was n=2? Could the low number of tested flies explain the irregular shape of the curve?In particular, between CT15 and CT18 there is a sudden jump to higher values of fluorescence intensity.Did this jump coincide with a lower number of flies tested?Would there be a possibility to color code the points so that one can immediately tell from how many flies was recorded?Lines 364-366: what do you mean with "a synaptic structure that indicates fast neurotransmitter D<:@4><@: H4D @AE 78E86E87 58EH88@ E;8 -/7D 4@7 0).#K& @8FCA@D <@ E;8 6A@@86EA?874E4J( ,@ the results section, you considered it very likely that these two neurons have synaptic contacts with each other and you showed this even via trans-Tango.Now you say that there are no synapses between the two types of neurons.Please clarify this discrepancy.Nevertheless, the discussed paracrine, modulatory input from the clock neurons is very likely, since all of them are neuropeptidergic.I wonder why you exclude a neuropeptidergic input to the 0).#K& @8FCA@D 9CA?E;8 D#-/GD G<4 0*+ 4@7%AC D/0+$ 3;8 9<58CD A9 E;8D8 EHA EIB8D A9 @8FCA@D are certainly close enough to enable paracrine signaling.At least for PDF this could be easily tested 5I 6).0#<?4:<@: <@ E;8 0).#K& @8FCA@D 7FC<@: E;8 4BB><64E<A@ A9 0*+$ +FCE;8C?AC8" E;8 BC8D8@68 A9 E;8 0*+#C868BEAC A@ E;8 0).#K& @8FCA@D 6AF>7 58 6;86=87 FD<@: 4 0*+1#:4>' ><@8$ 2<@68 IAF could prevent the loss of PAM neurons by expressing PER only in the PDF neurons (Figure 4), a D<:@4>><@: 9CA? E;8 0*+ @8FCA@D EA E;8 0).#K& @8FCA@D 4BB84CD G8CI ><=8>I$ 3;<D <D 46EF4>>I E;8 simplest explanation for your findings.
Lines 372-388: The bimodal Ca2+ rhythms in the MB299-labelled neurons are very interesting.Similar bimodal Ca2+ rhythms have been found in ring neurons of the ellipsoid body neurons by Liang et al (2019, see above).These ring neurons are rhythmically modulated by DA neurons of the PPM3 cluster, which get neuromodulatory input from the PDF neurons.Thus, this situation strongly resembles the findings in your study and I do not understand why this is not discussed and the Liang et al (2019) paper is not even cited.
Lines 408-410: Lyons and Roman (2008 Learn Mem 16) have already shown that the circadian clock modulates short-term memory.Later, Chouhan et al (2015 Curr Biol 25) even showed that flies remember the time of day, an ability that requires the input from the circadian clock to memory centers in the mushroom bodies.Citing these papers may help to discuss the non-motor symptoms of PD.
Figure 8: The sudden death of all per0 mutants treated with H2O2 on day 31 is hard to understand.You state that this experiment was repeated twice.It is hard to believe that all flies died exactly at the same day in both experiments.How many flies are included in the survival assay?Have they been kept in just one single large food vial or several food vials?How often food was changed?I did not find any description of this experiment in Material & Methods.This description must be added, and I strongly recommend to repeat this experiment once more.
Lines 145-147: please change into: "…showed a significant los of PAM neurons as coompared to the control group…" Lines 172-173: Just for interest: do you have an explanation for why flies drink three to five times more H2O2 in DD than in LD?
Reviewer #3 (Remarks to the Author): Dorcikova et al lay out an interesting and exciting study examining links between circadian rhythm and loss of dopaminergic neurons as a potential mechanism occurring in Parkinson's.The manuscript is very well written and clear with not observable typos or missing details that I can see.
The study uses flies to initially recapitulate a loss of DA neurons in response to H2O2, noting a circadian influence in the effect depending on time of delivery of the insult.They then examine DA neuronal loss in flies mutant for circadian clock components, noting a loss of neurons in the per0 mutant and a timeless mutant.Given that the chemical insult is delivered by feeding, they then examine any role for starvation or dehydration and find no role.
The key population of neurons are the PAM group and they examine synaptic linkage between circadian neurons and PAM neurons and find connections, data that is bolstered by the Janelia Farm connectome data.A subpopulation of the PAM neurons is then genetically defined and these are examined for their resting calcium levels in a 24h period, with a dramatic change in the calcium rhythms altered in the Per0 mutant.This data is interesting, but more descriptive than mechanistic as it stands.The subpopulation of PAM neurons are associated with the mushroom body, and may regulate sleep.The paper then studies the effects of the H2O2 administration on sleep and finds an increase in nighttime sleep.I'm not so comfortable with the specificity of this experiment, as the H2O2 delivery is systemic, but the conclusions based on the loss of the subset of DA neurons -so the linkage is limited here.
I like this study a great deal and feel that it opens up a productive area of study.My concerns are with the linkage between the feeding and the effects, is ROS in either the DA neurons or the Pdf neurons eliciting these effects.I'm assuming that is the claim, but it would be good to link ROS, either the pdf or DA neurons more directly than the presence of increased MitoSOX detected ROS in the PAM neurons.I would suggest a rescue of the H2O2 excess in either the pdf neurons or the PAM neurons using UAS-catalase (or some other anti-oxidant transgene -UAS-Trx, UAS-GST?) to reduce ROS specifically in these neurons to define whether ROS toxicity is causing this effect in pdf neurons, or DA neurons.
Overall though, this is a meticulous and novel study and potentially lays out mechanisms for the two-hit theory of insult causing PD The authors have significantly improved their manuscript, responding to most of my concerns.There is however a couple of issues that linger.Both are related to the functional consequences of H2O2 treatment.
1) The authors attempted to address my first major comment by inhibiting synaptic transmission in PAM1a neurons.The sleep phenotype observed with Shi-ts partially differs from that observed with H2O2 phenotype, which raises questions as to whether the loss of these neurons is really responsible for the H2O2 sleep phenotype.The authors propose that perhaps only partial inhibition occurred, or that the varying observations are the result of using different across experiments.Both are possible of course, but I wonder why the authors did not simply measure sleep after H2O2 treatment at 29C to avoid the potential confound of temperature.Also, the authors might have been able to ablate the neurons with HID, or completely suppress them with KIR.Finally, the authors did not attempt to determine whether H2O2 and PAM1a inactivation (even if partial) have additive effect or not, as I had suggested.They did not explain why.Thus, whether the sleep defect after H2O2 results from the loss of PAM1a neurons remain unclear.At the very least, the authors need to acknowledge that clearly.
2) Reviewer #2 had concerns with the sudden death observed around day 35 after H2O2 treatment of per0 flies.The authors added new data to figure 8C-D, but I have a hard time understanding what was done, based on how they responded to the reviewer's comments and the survival curves themselves.Are the curves now shown based solely on the new data, or do they combine old and new data?If the data now shown are all new, then there is still quite a bit of flies dying around day 35.This sudden death would be reproducible, though with variable amplitude.I would then suggest to show both the new and old data (the latter in a supplemental figure).This reproducible death around day 35 would clearly indicate an important impact of per on survival after H2O2 treatment.However, if the survival curves now shown are combining old and new data, then there seems to be a significant problem.Indeed, the only difference in the per0 curves would be coming from the death that occurred at day 35 in the first two experiments (rate of death are otherwise quite similar over time).Indeed, comparing the curves now shown to the old ones, I am guessing that the massive day-35 mortality was not observed in the two most recent experiments.This lack of reproducibility would be problematic Reviewer #2 (Remarks to the Author): In my opinion, manuscript is carefully revised and I have no further comments.
Reviewer #3 (Remarks to the Author): I feel that the authors have addressed all the points made by all the reviewers openly and honestly.I'm happy with the result.
72: citing the review of Hall from 2003 and an own review from 2019 in respect of the clock neurons and the clock network is certainly fine, but what about the recent work of Shafer et al. (2022, eLife 11) and Reinhard et al. (2022, JCN 530 and Front Physiol 19) that describe the clock network and their synaptic connections in greater detail?This work needs to be cited.Lines 72-74: again, only the authors' own work is cited.What about the work of Liang et al. (2019, Neuron 102) showing that the the circadian clock controls ring neurons in the ellipsoid body via PPM neurons?Since PPM neurons are also dopaminergic neurons, this work is very relevant to the present study (see also later).

Figure 2 :
Figure 2: Are the images in 2A superpositions of several confocal stacks or do they show only one confocal image?How thick are the individual confocal images?The image of the 1-day-old per0 fly appears to contain fewer stinger-positive PAM neurons than the images of the older flies, but based on the quantification in Fig.2C, this is not representative.The many small symbols (data points) in the box plots make it hard to distinguish the white (w1118) and gray (Canton-S) plots.Please try to optimize the pictures.In my opinion, it is enough to indicate the number of analyzed brain hemispheres below the box plots.